Supercoiling is a structural feature of topologically constrained DNA molecules, such as circular chromosomes and linear chromosomes whose ends are not free to rotate. In both prokaryotes and eukaryotes, regulation of DNA topology is fundamental to the viability of the cell. The long-term goal of the proposed research is to understand the relationship between DNA topology and the metabolic activities of the DNA molecule. The objective of this proposal is to determine the molecular basis of sequence-dependent differences in supercoiling among DNAs isolated from topoisomerase I mutants of bacteria. Recombinant DNA methodology and a high resolution, gel electrophoretic method of assaying supercoiling will be used to analyze the case of the pBR322-tetA gene, whose presence in cis greatly alters plasmid DNA supercoiling in topoisomerase I mutants. Both an intact promoter and a portion of the remainder of the gene, but not the gene product, are required for the effect of tetA on supercoiling. No particular section of the gene outside of the promoter appears to be necessary; only the size of the section remaining appears to be important, suggesting that transcription of tetA may be responsible for the gene's effect on supercoiling. The proposed research will identify what intrinsic features of the gene or features of pBR322 organization affect supercoiling in topoisomerase I mutants and will test the hypothesis that transcription of tetA is responsible for the effect. Physiological and biochemical studies will be initiated to identify the molecular interactions leading to the altered supercoiling distribution. This work should help to understand the interactions involved in regulating DNA supercoiling and lead to an increased understanding of the in vivo role(s) of topoisomerase I.